Materials and methods for intracellular delivery of biologically active molecules

ABSTRACT

The subject invention finds utility in the area of gene therapy of diseases. More specifically, the invention concerns the making of a novel non-viral vector which can bind to desired DNA to form a combination useful to transfect diseased mitochondria of human or animal cells. The non-viral vector comprises a dequalinium salt subjected to standard liposome production procedures to obtain the vector named DQAsomes.

The subject invention was made with government support under a researchproject supported by NIH Grant Nos. RO 1-GM-47535; R29-HL55770-02 andPO1-AG10485-06. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Since the first demonstration in 1988 that mitochondrial DNA (mtDNA)base substitution and deletion mutations are linked to human disease, avariety of degenerative diseases have been associated with mtDNAmutations (reviewed in Wallace, D. C. [1994] J. Bioenergetics andBiomembranes 26:241-250). For example, certain deleterious basesubstitutions can cause familial deafness and some cases of Alzheimer'sdisease and Parkinson's disease. Other nucleotide substitutions havebeen associated with Leber's Hereditary Optic Neuropathy (LHON) andmyoclonic epilepsy and ragged-red fiber disease (MERF). Basesubstitutions can also cause pediatric diseases such as Leigh's syndromeand dystonia. Severe rearrangements involving deletions have been linkedwith adult-onset chronic progressive external ophthalmoplegia(CPEO) andKearns-Sayre syndrome (KSS) as well as the lethal childhood disorderPearson's marrow/pancreas syndrome (Wallace [1994], supra).

Somatic gene therapy. Three different approaches for somatic genetherapy (reviewed in Ledley, F. D. [1996] Pharmaceutical Res. 13:1996)can be distinguished based on the nature of the material that isadministered to the patient: (a) cell-based approaches involving theadministration to the patient of genetically engineered cells("ex-vivo"), (b) administration to the patient of geneticallyengineered, attenuated, or defective viruses, and (c) plasmid-basedapproaches that involve pharmaceutical formulations of DNA molecules. Avariety of viral and non-viral methods have been developed forintroducing DNA molecules into a cell. Non-viral techniques includeprecipitation of DNA with calcium phosphate (Chen, C., H. Okayama [1987]Mol. Cell. Biol. 7:2745-2752), dextran derivatives (Sompayrac, L., K.Danna [1981] PNAS 12:7575-7584), or polybrene (Aubin, R. J., M.Weinfield, M. C. Paterson [1988] Somatic Cell Mol. Genet. 14:155-167);direct introduction of DNA using cell electroporation (Neuman, E., M.Schaefer-Ridder, Y. Wang, P. H. Hofschneider[1982] EMBO J. 1:841-845) orDNA microinjection(Capecchi, M. R. [1980] Cell 22:479-486); complexationof DNA with polycations (Kabanov, A. V., V. A. Kabanov [1995]Bioconjugate Chem. 6:7-20); and DNA incorporation in reconstructed viruscoats (Schreier, H., R. Chander, V. Weissig et al. [1992] Proceed.Intern. Symp. Control. Rel. Bioact. Mater. 19:70-71; Schreier, H., M.Ausborn, S. Guinther, V. Weissig, R. Chander [1995] J. Molecular Recog.8:59-62).

Cationic lipids have become important reagents for gene transfer invitro and in vivo. Several clinical trials approved by the NIH are inprogress (reviewed in Ledley, F. D. [1994] Current Opinion inBiotechnology 5:626-636; and Ledley, F. D. [1995] Human Gene Therapy6:1129-1144). In terms of transfection efficiency, virus-based vectorsare superior to all other DNA transfection methods. Several differentviral vectors have been developed and are in clinical trials includingthose derived from murine leukemia viruses (retroviruses),adeno-associatedvirus, and adenovirus (reviewed in Ledley [1996],supra).

Transfection of mitochondria. There have been only a few reports ofnucleic acids entering mitochondria, and most have focused on thenuclear encoded RNA component of the mitochondrial RNA processingactivity, RNase MRP (Chang, D. D., D. A. Clayton [1987] Science235:1178-1184; and Li, K., C. S. Smagula, W. J. Parsons et al. [1994] J.Cell. Biol. 124:871-882). The uptake of exogenous DNA into mitochondriainvolving the protein import pathway has been reported from twolaboratories. Vestweber and Schatz ([1989] Nature (London) 338:170-172)achieved uptake of a 24-bp both single- anddouble-strandedoligonucleotide into yeast mitochondriaby coupling the 5'end of the oligonucleotide to a precursor protein consisting of theyeast cytochrome c oxidase subunit IV presequence fused to a modifiedmouse dihydrofolate reductase. More recently, Seibel et al. (1995,Nucleic Acids Research 23:10-17) reported the import into themitochondrial matrix of double-stranded DNA molecules conjugated to theamino-terminal leader peptide of the rat ornithine-transcarbamylase.Both studies, however, were done with isolated mitochondria notaddressing the question of how oligonucleotide-peptide conjugates willpass the cytosolic membrane and reach mitochondrial proximity.Negatively-charged biological cell surfaces and lysosomal degradationestablish major obstacles which are very unlikely to be overcome bysingle oligonucleotide-peptide complexes.

Dequalinium. Dequalinium (DQA) (Babbs, M., H. O. J. Collier, W. C.Austin et al. [1955] J. Pharm. Pharmacol. 8:110-119)has been used forover 30 years as a topical antimicrobial agent. There is no consensusabout the molecular target of DQA; several different targets such as thesmall conductance Ca²⁺ -activated K⁺ channel, F1-ATPase, calmodulin, andproteinase K have been suggested (Dunn, P. M. [1994] Eur. J Pharmacology252:189-194; Zhuo, S., W. S. Allison [1988] Biochem. Biophys. Res. Comm.152:968-972; Bodden, W. L., S. P. Palayoor, W. N. Hait [1986] Biochem.Biophys. Res. Comm. 135:574-582; Rotenberg, S. A., S. Smiley, M. Ueffinget al. [1990] Cancer Res. 50:677-685). DQA is an amphiphilic dicationiccompound resembling bolaform electrolytes, that is, they are symmetricalmolecules with two charge centers separated at a relatively largedistance. Lipophilic cations are known to localize in mitochondria ofliving cells as a result of the electric potential across themitochondrial membrane (Johnson, L. V., M. L. Walsh, B. J. Bockus, L. B.Chen [1981] J. Cell. Biol. 88:526-535). The accumulation of DQA inmitochondria has been reported (Weiss, M. J., J. R. Wong, C. S. Ha etal. [1987] PNAS 84:5444-5448; Christman, E. J., D. S. Miller, P. Cowardet al. [1990] Gynecol. Oncol. 39:72-79; Steichen, J. D., M. J. Weiss, D.R. Elmaleh, R. L. Martuza [1991]J. Neurosurg. 74:116-122; Vercesi, A.E., C. F. Bernardes, M. E. Hoffman et al. [1991]J. Biol. Chem.266:14431-14434).

Despite the progress being made in developing viral and non-viral DNAdelivery systems, there is a need for an efficient method forintroducing DNA into mitochondria of intact cells.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to materials and methods for selectivelyand specifically delivering biologically active molecules to themitochondria. In a preferred embodiment, DNA or other polynucleotidesequence can be delivered to the mitochondria as part of a gene therapyprocedure.

The subject invention pertains to the delivery to the mitochondria of acomplex of DNA with a molecule having two positive charge centersseparated by a hydrocarbon chain. In a specific embodiment, the subjectinvention concerns the transformation of a salt of dequalinium (DQA)into an effective non-viral gene therapy vector. DQA is complexed withDNA as described herein to form an effective vehicle for delivering DNAto the mitochondria. These DQA-DNA complexes are referred to herein asDQAsomes. The DQAsomes can be used effectively as described herein as atransfection system. This system is especially useful in gene therapy totreat diseases associated with abnormalities in mitochondrial DNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the production of DQAsomes and the interaction with plasmidDNA. The DQAsomes can be produced utilizing standard liposome methods inconjunction with the teachings provided herein.

FIG. 2 shows the interaction of DNA and DQAsomes. This interaction isshown using a fluorescence-SYBR green method. In this procedure adecrease in fluorescence intensity is indicative of DNA/DQAsomeinteraction.

FIG. 3 shows the expression of reporter gene, firefly luciferase,measurable at an approximately equal mass ratio of DNA to dequalinium,corresponding to 72 μm DQAsomes.

FIG. 4 shows a comparison of the transfection efficiency betweenDQAsomes and DOTAP.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention provides materials and methods useful indelivering biologically active molecules to mitochondria. In a preferredembodiment, the subject invention provides a method for selectivelytransforming mitochondrial DNA. This method can be used to correctdefects in mitochondrial DNA.

In a specific embodiment, the subject invention pertains to the use ofan amphiphilic dicationic compound complexed with DNA to deliver the DNAspecifically to the mitochondria. In a preferred embodiment, theamphiphilic dicationic compound is a salt of dequalinium (DQA). The saltmay be, for example, dequalinium chloride (available from Sigma ChemicalCompany, St. Louis, Mo.). Using standard liposome production procedures,combined with the teachings provided herein, dequalinium chloride can betransformed into an effective non-viral gene therapy vector (DQAsomes).This is a novel use for DQA. This is the first disclosure that DQAsomesare effective as a transfection system.

The gene therapy vectors of the subject invention can be used to treatdiseases associated with mitochondrial DNA, for example, Alzheimer'sdisease, Parkinson's disease, Leber's Hereditary Optic Neuropathy,myoclonic epilepsy and ragged-red fiber disease, Leigh's syndromedystonia, adult-onset chronic progressive external ophthalmoplegia,Kearns-Sayre syndrome and Pearson's marrow/pancreas syndrome. The DNAdelivery vectors of the subj ect invention are particularly advantageousbecause these amphipathic dicationic compounds will specifically deliverDNA to the mitochondria. Thus, in a specific embodiment of the subjectinvention, DQAsomes can be used as a mitochondria-specificpolynucleotide delivery system.

Those skilled in the art, having the benefit of the instant disclosure,will appreciate that other salts of dequalinium can be used. In onespecific embodiment, dequalinium acetate (Sigma Chemical Company, St.Louis, Mo.) can be used. Other amphiphilic dicationic compounds whichcan be used according to the subject invention include all derivativesof dequalinium with varying substituents at the aromatic ring systemsincluding 1-1'-Decamethylene bis-quinolinium-salts, which have nosubstituents at all. The critical characteristics of the compounds whichcan be used according to the subject invention include the presence oftwo positive charge centers separated by a relatively long hydrocarbonchain. The hydrocarbon chain may have, for example, from about 5 toabout 20 carbons. In a preferred embodiment, the hydrocarbon chain mayhave from about 8 to about 12 carbons.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Preparation of DOAsomes

Dequalinium chloride: 0.1 mmol (53 mg) is dissolved in 20 ml of methanolin a 100-ml round bottom flask. The methanol is removed by the use of arotary evaporator at elevated temperatures (40° C.) resulting in a thin,well-dispersed film in the bottom of the flask. Sterile water (10 ml) isthen added to the flask and sonicated with a probe sonicator (power)until the mixture is clear. This results in a 10-mM dispersion of thedequalinium chloride in water, a product we have termed DQAsomes. SeeFIG. 1.

EXAMPLE 2 DOAsomes Binding of Plasmid DNA

Plasmid DNA (pGL3 luciferase firefly with SV-40 promoter, Promega) wasincubated with increasing amounts of DQAsomes for 30 minutes at roomtemperature to allow the binding of DNA to DQAsomes. Thereafter, "SYBRGREEN 1" (FMC) was added. The fluorescence was read 30 minutes later ona PE LS50B spectrometer with excitation at 497 nm, emission at 520 nm,and slit width 5 cm.

To assess the binding of DNA to DQAsomes the DNA specific dye "SYBRGREEN 1" was used. The fluorescent signal of this dye is greatlyenhanced when bound to DNA; non-binding results in loss of fluorescence.As can be seen in FIG. 2, DQAsomes strongly interact with plasmid DNA.Increasing amounts of DQAsomes prevent "SYBR GREEN 1" from binding tothe DNA, leading to a complete loss of the fluorescence signal.

EXAMPLE 3 Transfection of Cells Using DOAsomes as a Vector

Transfection of LLPKC 1 cells: cells were grown to 75% confluence inRPMI serum media with antibiotics before transfection. Transfectionmixtures contained non-serum media and pDNA/liposome mixtures, whichwere allowed to sit for 30 minutes before use. As a model fortransfection, plasmid DNA pGL3 luciferase firefly with SV-40 promoter(from Promega) was used. Each well received 15 μg of DNA and theappropriate amount of DQAsomes (total reaction volume 0.5 ml). Serum wasremoved and replaced with non-serum media. Cells were then incubated forone day before being washed with PBS and lysed with luciferase lysisbuffer. Expression of the reporter gene was measured with a Moonlightluminometer, and protein was determined with a Pierce protein assay kit.

The expression of the reporter gene firefly luciferase became measurableat an approximately equal mass ratio of DNA to dequalinium,corresponding to 72 μμM DQAsomes (FIG. 3). Doubling the amount ofDQAsomes further increased the expression, whereas at a mass ratio ofdequalinium to DNA of 1:4, the expression was drastically decreased.These results clearly demonstrate intracellular DNA delivery usingDQAsomes as a vector.

EXAMPLE 4 Comparison of the Transfection Efficiency Between DQAsomes andDOTAP

The transfection of LLPKC 1 cells was done as disclosed in Example 3.FIG. 4 shows a comparison of the transfection efficiency betweenDQAsomes and the widely-used DOTAP. The DQAsomal system has atransfection efficiency in the range of the commercially available DOTAPvector.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

What is claimed is:
 1. A complex comprising DNA and a molecule whichcomprises two positive charge centers separated by a hydrocarbon chainwherein said hydrocarbon chain has about 8 to about 20 carbons andwherein said molecule with two positive charge centers is a salt ofdequalinium.
 2. The complex, according to claim 1, wherein said salt isselected from the group consisting of the acetate salt and the chloridesalt.
 3. The complex, according to claim 1, wherein said DNA is plasmidDNA.